Dynamic Cell Division and Growth During de Novo Meristem Formation in Epiphytic Fern Gametophytes.

Meristems in land plants serve conserved functions, maintaining undifferentiated cells and continuously producing new cells to support organ development and body formation. In seed plants, de novo meristem formation involves the initiation and assembly of new stem cell niches, promoting organogenesis and shaping plant architecture. However, the function and regulation of de novo meristem formation in seed-free vascular plants remains largely unknown. Unlike seed plants, which have highly reduced gametophytes within a sporophyte-dominated life cycle, fern gametophytes develop pluripotent meristems that sustain growth independently of their sporophytes. Unlike the gametophytes of terrestrial ferns, epiphytic ferns exhibit programmed clone-forming or clonal growth during the gametophyte phase, suggesting enhanced or prolonged meristem activity. Using confocal live imaging with computer-assisted image segmentation and quantifications at single-cell resolution, we show that in Phlebodium pseudoaureum, a representative epiphytic fern, continuous de novo meristem formation drives gametophytic clone-forming and extended longevity. Patterns of cell growth and division are closely associated with the initiation and expansion of de novo-formed meristems in Phlebodium gametophytes, with active proliferation in the outermost layer driving this process. Our findings uncover the cellular basis of de novo meristem formation in epiphytic ferns and highlight both conserved and lineage-specific mechanisms controlling meristem initiation and proliferation in land plants.